Cosmetic or dermatological use of 7-hydroxylated steroids

ABSTRACT

A method of preventing the cutaneous effects of aging and/or the effects of UV irradiation on the skin, said method comprising applying to the skin a composition comprising at least one compound corresponding to the formula:  
                 
 
     in which:  
     R 1  is selected from: a hydrogen atom, organic acid ester of 1 to 24 carbon atoms, sulfuric ester or phosphoric ester functions, or carbon-containing ether of 1 to 24 carbon atoms optionally containing one or more nitrogen atoms, carbohydrate ethers of 3 to 100 carbon atoms optionally containing one or more nitrogen atoms;  
     R 2  is selected from: a hydrogen atom or a fatty acid ester function of 1 to 24 carbon atoms;  
     R 3  is selected from: a hydrogen atom, an —OH group, the groups of formulae:  
     —CO—R 4 , —CHOH—R 4 , ═CH—CH 3 , ═COH—CH 3 , —CHR 4 —CH 3 , ═O, in which R 4  is an alkyl group comprising 1 to 10 carbon atoms, which is substituted or unsubstituted.

[0001] The present invention relates to the use of 7-hydroxylatedsteroids for the preparation of cosmetic or dermatological compositionsfor preventing and/or treating the cutaneous effects of aging and of theaction of ultraviolet irradiation.

[0002] The formation of steroid hormones, their interrelations and theirfunctions have been widely described in the prior art. The functions ofpregnenolone (PREG) and of dehydroepiandrosterone (DHEA) as well as ofcertain of their derivatives are especially mentioned in the PCT patentapplication published under the number WO 94/08588.

[0003] DHEA and its sulfate derivative (S-DHEA) circulate in asignificant quantity in adult men, but its level decreases with age(Orentreich et al., J. Clin. Endocr. Metab. 59: 551-555, 1984). It wasthus proposed, for example in the French patent application publishedunder the number 2 729 854 or the corresponding European patentapplication published under the number 723 775, to use S-DHEA in acosmetic composition for topical application which is intended for thetreatment of certain signs of aging. Multiple effects of DHEA have beendescribed, but some of them oppose the processes and the pathologiesassociated with aging (Watson et al., Drug & Aging 9: 274-291, 1996).

[0004] Despite numerous experiments, it has not been possible to proveany of the explanations advanced for the effects of DHEA (Kalimi et al.,Molec. Cell. Biochem. 131: 99-104, 1994), and the therapeutic use ofDHEA has revealed undesirable secondary effects, in particular in women,as a potential precursor of androgenic hormones.

[0005] It has now been shown that the 7-hydroxylated derivatives of PREGand of DHEA are formed by an enzymatic system present in numeroustissues and organs, including the skin, where they favor the mechanismsconnected with immunity (Morfin & Courchay, J. Steroid Biochem. Molec.Biol. 50: 91-100, 1994). Like the levels of circulating DHEA, theactivity of these hydroxylating enzymes decreases with age (Doostzadeh &Morfin, Steroids 61: 613-620, 1996).

[0006] The Applicant is therefore interested in the effects of7-hydroxylated steroids and their derivatives on cells which form thehuman skin and which are affected during aging or after UV irradiation.The research work carried out by the Applicant has allowed it to bedemonstrated that the effects of glucocorticoids leading to cellapoptosis are cancelled out by the 7-hydroxylated steroids and thattheir action on the cutaneous cells is manifested by beneficial andprotective effects.

[0007] Surprisingly, the results obtained with the compounds of theinvention do not correspond to those conventionally expected withsteroid hormones. Indeed, the hydroxylation process carried out by thebody on PREG or DHEA is irreversible, and therefore the conventionalsteroid hormones cannot be produced from 7-hydroxylated derivatives.

[0008] Consequently, the use of 7-hydroxysteroids for cosmetologicalpurposes for treating or preventing the cutaneous effects of aging hasoutstanding advantages with respect to the steroids of the cosmeticcompositions of the prior art.

[0009] The recent work concerning the cutaneous modifications caused byage or UV and their medical treatment specifically envisages retinoicacid, α-hydroxy acids and DHEA, but does not mention 7-hydroxysteroids(Gilchrest, Brit. J. Dermatol. 135: 867-875, 1996; Watson et al., Drugs& Aging 9: 274-291, 1996).

[0010] The production of 7-hydroxylated derivatives of DHEA has beenknown for a long time in the tissues of the human fetus (Sulcova et al.,Endocr. Experiment. 2: 167-172, 1968), and in the amniotic epithelium(Sulcova et al., J. Steroid Biochem. 7: 101-104, 1976), the human liver(Starka, Sond. Zeit. Natur. 17: 1-2, 1965), human testicles andepididymis (Sulcova & Starka, Experimentia 28: 1361-1362, 1972) and inhuman pre-adipocytes (Khalil et al. J. Steroid Biochem. Molec. Biol. 46:585-594, 1993). In addition, the circulating levels of 7α-hydroxy-DHEAhave been measured in premenopausal women at 200-300 pg/ml (Skinner etal. Steroids 30: 315-330, 1977), and 3β,7α-dihydroxy-5α-androstan-17-one(7α-dihydroxyisoandrosterone) has been characterized in human urine(Jacolot et al. J. Steroid Biochem. 14: 663-669, 1981). More recently,the phenomenon of 7-hydroxylation has been extended to other steroidswhich have, in common with DHEA, a 3β-hydroxylated structure. These arePREG (Akwa et al. Biochem J. 288: 959-964, 1992; Morfin & Courchay J.Steroid Biochem. Molec. Biol. 50: 91-100, 1994), 5α-androstane-3β,17β-diol (Morfin et al. Biochimie. 59: 637-644, 1977; Morfin et al. J.Steroid Biochem. 12: 629-632, 1980), 3β-hydroxy-5α-androstan-17-one(Akwa et al. Biochem. J. 288: 959-964, 1992) and3β-hydroxy-5α-pregnan-20-one (Stromstedt et al. Molec. Pharmacol. 44:1077-1083, 1993).

[0011] Some work on 7-hydroxylated steroids proved that they werewithout characteristic hormonal effects of both androgenic andestrogenic type or on the secretion of pituitary hormones (Celotti etal. J. Steroid Biochem. 18: 397-401, 1983; Sunde et al. J. SteroidBiochem. 16: 483-488, 1982). All of these results therefore lead to the7-hydroxylation of steroids being considered as a terminal process ofhormonal inactivation leading to the urinary and biliary excretion ofthe 7-hydroxylated steroids produced (Ofner et al. J. Steroid Biochem.11: 1367-1379, 1979; Stromstedt et al. Molec. Pharmacol. 44: 1077-1083,1993; Khalil et al. J. Steroid Biochem. Molec. Biol. 48: 545-552, 1994).It is only very recently that it has been possible in part to explainthe multiple effects noted with DHEA (Watson et al. Drug & Aging 9:274-291, 1996) by the immunostimulatory properties of its 7-hydroxylatedderivatives (Morfin & Courchay J. Steroid Biochem. Molec. Biol. 50:91-100, 1994; Padgett & Loria J. Immunol. 153: 1544-1552, 1994; Loria etal. J. Endocrinol. 150: S209-S220, 1996). The antiglucocorticoidproperties shown by 7α- and 7β-hydroxy-DHEA have been proved andextended to other 7-hydroxylated steroids like those described in thePCT patent applications published under the numbers WO 93/20687 and WO94/08588 for their role in the triggering of immune processes.

[0012] It therefore appears that DHEA and the production of its7-hydroxylated derivatives decrease with age although that of theglucocorticoids does not vary. In the course of aging, the contributionof hormonal steroids to the cutaneous level is therefore found to bemodified with a predominance of glucocorticoids whose promoter effectson cutaneous aging are known.

[0013] Consequently, a localized contribution of 7-hydroxylated steroidsendowed with a particular but natural antiglucocorticoid effect allowsthe treated skin to be restored to its youthful steroid context.

[0014] However, these properties have never been described or suggestedin the prior art. Thus, the PCT patent application published under thenumber WO 94/08588 does not describe or teach any cosmetic ordermatological application of steroid hormone derivatives. In addition,this patent application is directed at steroidal derivatives in whichthe substitutions in positions 3 and 7 indicated in the formula (I)below are either hydroxyls or ester functions of 1 to 10 carbon atoms.

[0015] The European patent application published under the number 415766 describes the use of retinoid agents for combating cutaneous atrophyby an antiglucocorticoid mechanism. However, no structural relationshipexists between the retinoids (vitamin A and its derivatives) and thesteroids.

[0016] The European patent application published under the number 189738 describes the use of dehydroepiandrosterone (DHEA) and of its esterderivatives for treating drying out of the skin, but these compounds aredifferent from the steroids which are the subject of the presentinvention.

[0017] The European patent application published under the number 723775 envisages the use of the sulfate of DHEA in cosmetic anddermatological compositions and suggests the addition to thesecompositions of steroid hormones other than the sulfate of DHEA, such asandrogens, estrogens and progestagens. However, as indicated above,these steroids are without hormonal action, and their use only has theaim of alleviating the undesirable hormonal effects of DHEA and of thesulfate of DHEA. In addition, the sulfate of DHEA has no structuralrelationship with the steroids which are the subject of the presentinvention.

[0018] The invention therefore relates to the use, in a cosmetic ordermatological composition for topical application intended to preventor treat the symptoms of cutaneous aging and/or the effects of UVirradiation on the skin, of a 7α- or 7β-substituted compound of DHEA orof PREG, which is or is not reduced in position 5, and thuscorresponding to the formula:

[0019] in which:

[0020] R₁ is selected from: a hydrogen atom, organic acid ester of 1 to24 carbon atoms, sulfuric ester or phosphoric ester functions, orcarbon-containing ether of 1 to 24 carbon atoms comprising zero or anumber of nitrogen atoms, carbohydrate ethers of 3 to 100 carbon atomsand their derivatives including those comprising or not comprising oneor a number of nitrogen atoms.

[0021] R₂ is selected from: a hydrogen atom or a fatty acid esterfunction of 1 to 24 carbon atoms.

[0022] R₃ is selected from: a hydrogen atom, an —OH group, the groups offormulae: —CO—R₄, —CHOH—R₄, ═CH—CH₃, ═COH—CH₃, —CHR₄—CH₃, ═O, in whichR₄ is an alkyl group comprising 1 to 10 carbon atoms, preferably methyl,which is substituted or unsubstituted.

[0023] The compounds of the invention are 7α- or 7β-substitutedderivatives of DHEA or PREG and more particularly still 7α- or7β-hydroxylated derivatives which are or are not reduced in position 5.

[0024] A preferred group of compounds of the invention are the7α-hydroxylated derivatives, that is to say those in which the oxygencarried in position 7 is axial (7α) and the substituent R₂ is ahydrogen.

[0025] Another group of preferred compounds of the invention are thosewhere R₁ is hydrogen, especially 7α-hydroxy-DHEA and7α-hydroxyisoandrosterone where R₃ is a ketone (═O).

[0026] It is expedient to observe that the derivatives of the inventionin which R₁ is an organic acid have an increased fat solubility whichoffers the advantage of improving the retention of these compounds inthe cells, especially at the level of membranes, and consequently ofprolonging their activity and their effect on the cutaneous cells. Amongthese derivatives, those are preferred in which R₁ is a palmitate, anoleate or a ferulate, and especially 3β-palmitoyl-7ξ-hydroxy-DHEA,3β-oleyl-7ξ-hydroxy-DHEA and 3β-feruloyl-7ξ-hydroxy-DHEA.

[0027] The cosmetic or dermatological compositions of the invention cancomprise or one or more steroid derivatives according to the invention,as well as other compounds known for their cosmetological ordermatological property, like hormones and, of course, the adjuvants orvehicles conventionally used in these fields.

[0028] For the use of a steroid derivative of the invention in acosmetic composition intended to compensate, treat and/or prevent thecutaneous effects of aging and/or the effects of UV irradiation on theskin, said derivative is administered at a dose of between 0.05 and 10mg per application and per day and preferably between 0.05 and 5 mg perapplication and per day.

[0029] The effect of restoration or of prevention of cutaneous aging inpeople of a certain age as well as protective effects with respect to UVis applicable for any treatment aiming to restore the cutaneous tone,tone up the skin and smooth out wrinkles.

[0030] On account of their nature, the derivatives of the invention canbe employed in very diverse pharmaceutical forms for their percutaneousadministration. These can be forms resulting from the addition to thederivatives of the invention of cosmetically acceptable compounds andallowing creams, pastes, gels, lotions, “water-in-oil” or “oil-in-water”emulsions as well as forms composed of liposomes of simple or mixedmicelles or other penetration promoters such as lysophospholipids,cyclodextrins, polyethylene glycol, surfactants, alcohols, fatty acidsand vegetable oils to be produced. This list is not limiting and anyother presentation known to man can be envisaged since it is adapted tothe steroid derivatives of the invention which have, as acharacteristic, the property of being water-soluble and fat-soluble atthe same time. Thus, the cosmetic or dermatological compositions of theinvention can be present in the form of creams, lotions, gels andointments or any other form generally used for topical applications.

[0031] Other advantages and characteristics of the invention will appearon reading the examples which follow, given in a non-limiting capacity,and showing the performances obtained by the derivatives of theinvention as antiapoptotic and anti-free radical agents and promoters ofthe proliferation of human cutaneous cells.

EXAMPLE 1 Effects of 3β,7α-dihydroxy-5-androsten-17-one(7α-hydroxy-DHEA) and of 3β,7α-dihydroxy-5α-androstan-17-one(7α-hydroxy-ISOA) on Cell Apoptosis Induced by Glucocorticoids

[0032] The thymus of C57BL/6 mice aged 4 weeks is removed. Culturing ofthe thymocytes is carried out for 6 hours in RPMI 1640 medium and in thepresence or in the absence of the steroid tested. Apoptosis(fragmentation of the DNA) is measured by flow cytometry after labelingwith propidium iodide. The apoptotic phenomenon is checked byelectrophoresis of the DNA, which is visualized by ethidium bromideaccording to the conventional technique (observation of ranges of 200base pairs). The results reported in Table I below were obtained: TABLEI Steroids in the medium Apoptotic (in 10 ml of ethanol) cells (%)Ethanol alone 41.5 Dexamethasone 10⁻⁶ M 72.7 Dexamethasone 10⁻⁶ M + DHEA10⁻⁶ M 39.0 Dexamethasone 10⁻⁶ M + 7α-hydroxy-DHEA 10⁻⁶ M 58.8Dexamethasone 10⁻⁶ M + 7α-hydroxy-ISOA 10⁻⁶ M 72.0 Dexamethasone 10⁻⁵ M73.5 Dexamethasone 10⁻⁵ M + DHEA 10⁻⁵ M 51.4 Dexamethasone 10⁻⁵ M +7α-hydroxy-DHEA 10⁻⁵ M 48.6 Dexamethasone 10⁻⁵ M + 7α-hydroxy-ISOA 10⁻⁵M 46.3

[0033] It appears from these assays that the 7α-hydroxysteroids testedhave an antiapoptotic effect opposed to that of dexamethasone on mouse Tcells. Their effect at 10⁻⁵M is greater than that of their precursorsteroid (DHEA or dehydroepiandrosterone or3β-hydroxy-5-androstene-17-one).

EXAMPLE 2 Effects of 3β,7α-dihydroxy-5-androsten-17-one(7α-hydroxy-DHEA) on the Viability of Human Keratinocytes in Culture

[0034] Human keratinocyzes are obtained from parts removed by surgeryand are cultured in a monolayer to preconfluence. 7α-Hydroxy-DHEA isadministered to these cultures at various concentrations in ethanolicsolution (10%), each concentration being tested eight times. Controlsare carried out with ethanol alone (10%). After 24 hours, the viabilityof the keratinocytes is measured by testing with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) where themitochondrial dehydrogenase succinate transforms the MTT into bluecrystals of formazan which are soluble in DMSO (Mosmann, J. Immunol.Methods 65: 55-63, 1983). The results of assays on the viability of thekeranocytes are reported in Table II below. The cell viability iscalculated according to the formula:

% viability=product OD ₅₄₀×100/control OD ₅₄₀.

[0035] Any value greater than 100 indicates a product favoring cellviability. TABLE II Steroids in the medium Viability of (in 10% ofethanol) keratinocytes (%) 10% of ethanol alone (control) 1007α-hydroxy-DHEA 10⁻⁴ M  124 ± 10 7α-hydroxy-DHEA 5.10⁻⁵ M 111 ± 77α-hydroxy-DHEA 10⁻⁵ M 119 ± 7 7α-hydroxy-DHEA 5.10⁻⁶ M 147 ± 97α-hydroxy-DHEA 10⁻⁶ M 154 ± 6 7α-hydroxy-DHEA 5.10⁻⁷ M 139 ± 37α-hydroxy-DHEA 10⁻⁷ M 147 ± 5 7α-hydroxy-DHEA 10⁻⁸ M 127 ± 3

[0036] These results show that 7α-hydroxy-DHEA significantly increasesthe viability of human keratinocytes at concentrations of between 10⁻⁴Mand 10⁻⁶M, the maximum (increases between 54% and 39% in the viability)being obtained between 5.10⁻⁶M and 10⁻⁷M. In addition, no cytotoxicitywas observed. Other comparative tests demonstrated that the DHEAprecursor was without effect (100±5).

EXAMPLE 3 Effects of 3β,7α-dihydroxy-5-androsten-17-one(7α-hydroxy-DHEA) on the Proliferation of Human Fibroblasts in Culture

[0037] The human fibroblast cultures (woman of 32 years) were inoculatedinto 24-well plates at a rate of 50,000 cells/well in standard culturemedium (DMEM, gentamycin, amphotericin B, penicillin, L-glutamine, 10%FCS). The assays are carried out on 4 series of 3 wells. After 24 h, thefibroblasts adhere to the support and 3 series are treated with7α-hydroxy-DHEA at concentrations of 10⁻⁶M, 5.10⁻⁶M and 10⁻⁷M. Thefourth series contains only the vector (ethanol) . The media are reneweddaily, and at 96 h (72 h contact of the 7α-hydroxy-DHEA in the assay),the fibroblasts are counted in Malassez cells in the presence of trypanblue.

[0038] The results of the effects on the proliferation of fibroblastsare reported in Table III below. TABLE III Increase in Number of theviability Steroids in the medium fibroblasts (%) Control 190 667 ± 6 776/ 7α-hydroxy-DHEA 10⁻⁷ M 230 667 ± 8 511 +21 7α-hydroxy-DHEA 10⁻⁶ M  268000 ± 27 154 +41 7α-hydroxy-DHEA 5.10⁻⁶ M 258 667 ± 3 351 +36

[0039] These results demonstrate that, under the experimentalconditions, the treatment of fibroblasts by 7α-hydroxy-DHEA at 10⁻⁷M,10⁻⁶M and 5.10⁻⁶M increases the cell proliferation by 21%, 41% and 36%respectively with respect to the nontreated control fibroblasts.

EXAMPLE 4 Anti-free Radical Effect of 3β,7α-dihydroxy-5-androsten-17-one(7α-hydroxy-DHEA) on a Suspension of Human Keratinocytes

[0040] Keratinocytes originating from a healthy donor (woman of 25years) are cultured to the subconfluent stage in medium (KGM) which isspecific for the proliferation of the keratinocytes. The suspensionsobtained are divided in triplicate into 4 series of which 3 areirradiated for 30 min with a lamp emitting UVA in order to activate theproduction of free radicals. Among the three irradiated series, onecontains the vitamins C+E (0.7%) and serves as a reference ofprotection, one contains 7α-hydroxy-DHEA at 10⁻⁶M and the last serves asa control. Table IV below reports the measurement of the anti-freeradical effects.

[0041] The free radicals produced generate lipid peroxides which aredetermined by chemiluminescence (Belghmi et al. J. Biolum. Chemilum. 2:113-119, 1982). The efficacy of 7α-hydroxy-DHEA is calculated on thebasis of nonirradiated controls and of the reference of protection.TABLE IV Keratinocytes Chemiluminescence Efficacy Nonirradiated controls2 529 ± 153 / Irradiated controls  427 750 ± 137 322 / Irradiated + 0.7%Vit. C + E 2 970 ± 288 100% Irradiated + 7α-hydroxy-DHEA 10⁻⁶ M  44 164± 13 303  90%

[0042] Under the conditions of this study the in vitro anti-free radicalefficacy of 7α-hydroxy-DHEA is 90% at 10⁻⁶M. 7α-Hydroxy-DHEA can beconsidered as a good anti-free radical product.

1. A method of preventing the cutaneous effects of aging, said methodcomprising applying to the skin a composition comprising at least onecompound corresponding to the formula:

in which: R₁ is selected from: a hydrogen atom, organic acid ester of 1to 24 carbon atoms, sulfuric ester or phosphoric ester functions, orcarbon-containing ether of 1 to 24 carbon atoms optionally containingone or more nitrogen atoms, carbohydrate ethers of 3 to 100 carbon atomsoptionally containing one or more nitrogen atoms; R₂ is selected from: ahydrogen atom or a fatty acid ester function of 1 to 24 carbon atoms; R₃is selected from: a hydrogen atom, an —OH group, the groups of formulae:—CO—R₄, —CHOH—R₄, ═CH—CH₃, ═COH—CH₃, —CHR₄—CH₃, ═O, in which R₄ is analkyl group comprising 1 to 10 carbon atoms, which is substituted orunsubstituted.
 2. The method of claim 1, wherein R₂ and/or R₁ is ahydrogen atom.
 3. The method of claim 1, wherein R₃ is a ketone.
 4. Themethod of claim 1, wherein R₁ is a fatty acid ester function selectedfrom an oleate, a palmitate or a ferulate.
 5. The method of claim 2,wherein the compound of formula (I) or (II) is a 7α-hydroxy-DHEA.
 6. Themethod of claim 3, wherein the compound of formula (I) or (II) is a7α-hydroxyisoandrosterone.
 7. The method of claim 4, wherein thecompound of formula (I) and (II) is selected from the group consistingof 3β-palmitoyl-7ξ-hydroxy-DHEA, 3β-oleyl-7ξ-hydroxy-DHEA and3β-feruloyl-7ξ-hydroxy-DHEA.
 8. The method of claim 1, wherein thecomposition contains at least one compound of formula (I) or (II)combined with one or more adjuvants or vehicles used in cosmetology ordermatology.
 9. The method of claim 1, which comprises applying to theskin a dose of said composition of between 0.05 and 10 mg perapplication and per day.
 10. The method of claim 1, which comprisesapplying to the skin a dose of said composition of between 0.05 and 5 mgper application and per day.
 11. The method according to claim 1,wherein the composition is a composition aiming to restore the cutaneoustone.
 12. The method according to claim 1, wherein the composition is acomposition aiming to tone up the skin.
 13. The method according toclaim 1, wherein the composition is a composition aiming to smooth outwrinkles.
 14. The method of claim 8, wherein the composition is in theform of a cream, lotion, gel or ointment.